Human plasma contains approximately 3000 proteins with a variety of functions and potential therapeutic uses. Tight control of plasma available for blood fractionation means that the supply of important therapeutic agents like IgG is severly curtailed. This together with methodology which ends in very low yields and takes three to five days contributes to the international shortfall of major plasma fractions.
The present inventors have found that rapid isolation times, high recoveries and high-resolution make Gradiflow™ technology a viable alternative purification technology to conventional Cohn precipitation and column chromatography [1, 2].
Albumin and IgG both have enormous importance in medicine and therefore are of considerable commercial value. Albumin alone has an estimated annual global market value of $US1.5 billion [3]. Conventional purification protocols are cumbersome and expensive with low yields and long processing times [4].
Albumin is the most abundant protein component (50 mg/mL) in human plasma and functions to maintain whole blood volume and oncotic pressure. Albumin also regulates the transport of protein, fatty acids, hormones and drugs [4]. Clinical uses include blood volume replacement during surgery, treatment of shock, serious burns and other medical emergencies and the stabilisation of other pharmaceutical products.
Albumin has a molecular mass of 67 kDa and an isoelectric point (pI) of approximately 4.9. The protein consists of a single subunit and is globular in shape [5]. Conventional purification schemes use the Cohn ethanol precipitation method and result in only 50% recovery.
Immunoglobulin G (IgG) is the most abundant of the immunoglobulins, representing almost 70% of the total immunoglobulin component in human serum. The concentration of IgG in normal plasma is approximately 10 mg/mL [6]. The IgG plays an essential role in the immune response and have clinical uses including treatment of snake and spider bites, neurological disorders and IgG is commonly used in analytical or diagnostic kits.
The gamma-globulins have a molecular mass of approximately 150 kDa and consist of four chains, two of which are light and two of which are heavy [6]. Immunoglobulins are traditionally isolated using Cohn ethanol precipitation or alternatively affinity chromatography [7].
Alpha-1-antitrypsin is an acid glycoprotein of 54 kDa with an isoelectric point of 4.8 and is used in the treatment of hereditary emphysema [8]. Conventional purification schemes utilise a combination of Cohn fractionation and column chromatography with the major difficulty being the removal of albumin from α-1-antitrypsin preparations [9]. Current production schemes provide a yield of approximately 30% and much of this is contaminated with albumin. The present inventors have adapted Gradiflow™ to provide an alternative technique for producing highly pure α-1-antitrypsin with a yield of above 70%. This strategy also exemplifies Gradiflow™ technology's use in isolating protease inhibitors.
Gradiflow™ Technology
Gradiflow™ technology utilises molecular characteristics of size and charge to isolate protein [1] with the resolution of two-dimensional electrophoresis and the throughput of preparative chromatography. Proteins exist as charged molecules above or below their isoelectric point (pI). In the Gradiflow™ the net charge on a macromolecule is controlled by the choice of buffer pH. The proteins are separated in an electric field by charge and/or size differences [2].
The present inventors have found that the Gradiflow™ technology can be adapted to purify a number of different biomolecular components from plasma. The present inventors have devised methodology for the rapid isolation of albumin, IgG and α-1-antitrypsin from a single volume of plasma in a four-phase process with high yield and low cost.